Zwitterionic dyes for labeling in proteomic and other biological analyses

ABSTRACT

The invention relates to compositions and methods useful in the labeling and identification of proteins. The invention provides for highly soluble zwitterionic dye molecules where the dyes and associated side groups are non-titratable and maintain their net zwitterionic character over a broad pH range, for example, between pH 3 and 12. These dye molecules find utility in a variety of applications, including use in the field of proteomics.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/623,447, filed Jul. 18, 2003 which claims the benefit of thepriority date of U.S. Ser. No. 60/396,950, filed Jul. 18, 2002, both ofwhich are hereby expressly incorporated by reference.

GOVERNMENT INTERESTS

This research was supported by the US National Science Foundation GrantMCB 0139957 and US National Institutes of Health Grant R21RR16240.

FIELD OF THE INVENTION

The invention relates to compositions and methods useful in the labelingand identification of proteins. The invention provides for highlysoluble zwitterionic dye molecules where the dyes and associated sidegroups are non-titratable and maintain their net zwitterionic characterover a broad pH range, for example, between pH 3 and 12. These dyemolecules find utility in a variety of applications, including use inthe field of proteomics.

BACKGROUND OF THE INVENTION

Proteomics is the practice of identifying and quantifying the proteins,or the ratios of the amounts of proteins expressed in cells and tissuesand their post-translational modifications, under differentphysiological conditions. Proteomics also encompasses the analysis ofprotein-protein interactions. Proteomics provides methods of studyingthe effect of biologically relevant variables on gene expression andprotein production that provides advantages over genomic studies. Whilefacile DNA chip methods have been rapidly developed and are widelyavailable for analysis of mRNA levels, recent studies have shown littlecorrelation between mRNA levels and levels of protein expression (Gygi,S. P., et al., (1999) Correlation between protein and mRNA abundance inyeast, Mol Cell Biol. 19, 1720-1730; Anderson, L., and Seilhamer, J.(1997) A comparison of selected mRNA and protein abundances in humanliver, Electrophoresis, 18: 533-537). Furthermore, the functional stateof a large fraction of proteins in cells is largely determined bypost-translational modification, which must be analyzed directly at theprotein level.

Proteomics can be performed using multiplex detection methods. Multiplexdetection, or multiplexing, is defined as the transmission of two ormore messages simultaneously with subsequent separation of the signalsat the receiver. Multiplex fluorescence methods include, for example,multi-color fluorescence microscopy, multi-color fluorescent DNAsequencing, and two-color cDNA/mRNA expression array “chips”. Thesetechniques have been applied most commonly to the fields of cell biologyand genomics. However multiplex fluorescence methods are also applicableto the field of proteomics. Current multiplex methods in use in thefield of proteomics suffer from lack of detection sensitivity (U.S. Pat.No. 6,043,025; Amersham/Biosciences Operation Guide (2003) Ettan DIGEsystem; Beaumont, M., et al., (2001), Integrated technology platform forfluorescence 2-D difference gel electrophoresis, Life Science News,March 2001; Yan, J. X., et al., (2002) Fluorescence 2-D Difference GelElectrophoresis and mass spectrometry based proteomic analysis ofEscherichia coli, Proteomics 2: 1682-1698; Orange, P., et al., (2000),Fluorescence 2-D difference gel electrophoresis, Life Science News 5,1-4; Patton W F, Beechem J M., (2002) Rainbow's end: the quest formultiplexed fluorescence quantitative analysis in proteomics, Curr OpinChem. Biol. 6(1):63-9.

Predictions of cellular proteins from genome sequences indicate that twodimensional gel electrophoresis (2DE), with narrow isoelectric focusingpH ranges and cellular subfractionation, has the ability to resolvemany, and sometimes essentially all, of the proteins in cells. However,the full potential protein detection potential of 2DE has not beenrealized primarily because of limitations in detection sensitivity andgel-to-gel reproducibility.

A major limitation of current proteomics techniques is the lack ofcompositions and methods that provide sufficient sensitivity to detectlow levels of proteins. For example, proteins present at low copy numberare difficult to detect using currently available methods that generallyrely on the use of dyes to label proteins. In general, the dye moleculescurrently used in the art for detection of proteins during proteomicanalysis possess a number of undesirable qualities. Notably, thepresence of available dyes bound to the proteins before separationresults in a substantial decrease in solubility of the proteins. Thisbecomes especially problematic during the use of certain techniques usedto separate the proteins, such as two-dimensional gel electrophoresis.Loss of protein solubility during the separation process results in lossof detectable proteins. With currently available techniques the lack ofsolubility increases as the number of dye molecules per protein moleculeincreases. Thus, one cannot counter the lack of dye sensitivity byadding more dye molecules to the protein. In addition, the addition ofdyes can alter the isoelectric points (pls) of the proteins, causingserious perturbations in the resolution of proteins using techniquessuch as 2DE, for example. Methods that relay on detecting proteins withdyes or other stains after separation suffer from lack of sensitivity,do not allow multiplex detection, and may have low dynamic range fordetection, such as when using silver staining.

Other currently available proteomic techniques involve the use ofbiosynthetic isotopic labeling (Oda, Y., et al., (1999) Accuratequantitation of protein expression and site-specific phosphorylation,Proc. Natl. Acad. Sci. U.S.A 96: 6591-6596). This method is not readilyapplicable to animals or tissues and also requires mass spectralcharacterization of all the proteins separated, since expressiondifferences are not apparent without analysis of the isotopic labels.Additional methods use predigestion of proteins into a large number ofpeptides before separation and derivatization of cysteine residues withisotope and affinity tags (Gygi, S. P., et al., (1999) Quantitativeanalysis of complex protein mixtures using isotope-coded affinity tags,Nat. Biotechnol. 17: 994-999.) or alternatively derivatization ofN-terminal or lysine groups and isotope and/or affinity tags.Predigestion of proteins before separation produces a vast number ofpeptides that must be separated and analyzed for every experiment, avery demanding analytical process that is often hard to fully reproduce.The vast number of peptides that must be separated makes it extremelydifficult to obtain high coverage of the protein sequences in theanalysis, and if cysteine labeling is used only a small fraction of thepeptides are analyzed. Thus it is very difficult to detectpost-translational modifications in a general and reliable way usingmethods that require digestion of proteins into peptides beforeseparation and analysis.

Thus a need exists for optical labeling molecules that possess enhancedproperties of increased sensitivity and solubility to enhance detectionsensitivity and recovery of intact proteins, to avoid perturbation ofprotein charge or isoelectric point, to allow versatile multiplexanalysis of intact proteins for proteomics, so that intact proteins ofinterest can be selected more effectively and isolated for in depthanalysis of post-translational protein modifications. In addition, thereis a need for high sensitivity fluorescent covalent labeling dyes thatare highly water soluble, that preserve the net charge of the labeledprotein over a wide pH range for other applications that can benefitfrom the use of dye-labeled proteins.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides optical labeling molecules comprising at least one zwitterionicdye moiety, a titratable group moiety, and a functional linker moiety.

In a further aspect of the invention, the optical labeling moleculefurther comprises a cleavable moiety.

In a preferred embodiment of the invention, the charges on thezwitterionic dye moiety of the optical labeling molecule are independentof pH or non-titratable.

In one embodiment of the invention, the linker of the optical labelingmolecule is an amine-reactive linker. In an additional embodiment of theinvention, the linker is a thiol-reactive linker. The linker may beselected from the group consisting of imidoesters, N-hydroxysuccinimidylesters, sulfhydryl-reactive maleimides, and iodoacetamides. Preferredlinkers include, but are not limited to, succinimidyl groups,sulfosuccinimidyl groups, imido esters, isothiocyanates, aldehydes,sulfonylchlorides, arylating agents, maleimides, iodoacetamides, alkylbromides, or benzoxidiazoles.

In yet a further aspect of the invention, the optical labeling moleculefurther comprises a second label. The second label can be, for example,a light stable isotope label or one or more heavy stable isotope labels.

In a preferred embodiment of the invention, the charges on thezwitterionic dye moiety of the optical labeling molecule are stablebetween pH 3-12.

In a preferred embodiment of the invention, the zwitterionic dye moietyof the optical labeling molecule comprises a BODIPY dye with at leastone zwitterionic component.

The optical labeling molecule may have one of the following generalstructures:

-   -   T-ZD-A-; ZD-T-A-; ZD-T-C-A; T-ZD-C-A-; T-ZD-C-I-A-; or        ZD-T-C-I-A-;    -   wherein ZD is a zwitterionic dye moiety, T is a titratable        moiety, C is a cleavable moiety, I is a stable isotope moiety        and A is a linker moiety.

A further aspect of invention provides for a target protein labeled withan optical labeling molecule of the invention, wherein the linker of theoptical labeling molecule is covalently attached to the target protein.

In an additional aspect, the invention provides for a method of labelinga target protein comprising the steps of providing an optical labelingmolecule comprising a zwitterionic dye moiety, a titratable groupmoiety, an optional cleavable moiety, and a functional linker moiety andcontacting the target protein with the labeling molecule to form alabeled protein.

In yet a further aspect of the invention, a plurality of target proteinsare each labeled with a different optical labeling molecule of theinvention.

In an additional aspect, the invention provides for a method ofperforming protein analysis on a plurality of proteins comprisingproviding a plurality of different labeled proteins, each comprising adifferent zwitterionic dye moiety, a titratable group moiety, and anoptional cleavable moiety, and determining the presence or absence ofeach of the different labeled proteins.

In yet a further aspect, the invention provides for the additional stepswherein the plurality of different labeled proteins are mixed andseparated simultaneously prior to the determining the presence orabsence of each of the different labeled proteins in the samples. Thedifferent labeled proteins may be separated by a method selected fromthe group consisting of 1D gel electrophoresis, 2D gel electrophoresis,gel electrophoresis, capillary electrophoresis, 1D chromatography, 2Dchromatography, 3D chromatography, and the identities of the proteinsidentified by mass spectroscopy.

A further aspect of the invention provides for a method of proteinanalysis further comprising the step of determining the relativequantity of the different labeled proteins.

In yet a further aspect, the invention provides for a method of proteinanalysis wherein the cleavable moiety is present on the optical labelingmolecule, the method further comprising cleaving the cleavable moiety toremove the labeling molecule from the different labeled proteins. In afurther embodiment, the identities of the proteins separated by theabove method and their post-translational modifications are determinedby mass spectral techniques after removal of the dye tags.

An additional aspect of the invention provides for a method as describedabove wherein the cleavable moiety is present on the optical labelingmolecule and each of the labeled proteins further comprise a differentstable isotope tag moiety located between the functional linker moietyand the cleavable moiety. A further aspect provides for the additionalsteps of cleaving the cleavable moiety to produce isotope labeledproteins. A further aspect of the invention provides for thedetermination of the identity and post-translational modifications ofthe isotope labeled proteins by mass spectral techniques.

An additional aspect of the invention provides for a method of makingthe optical labeling molecules of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depict a number of suitable schematic configurations for theaddition of zwitterionic groups to dyes (1A, 1B and 1C) and dyederivatives (1D and 1E). A number of dye chromophores can be used andmodified to embody the essential aspects of this invention

FIG. 2 depicts the general structure of the class of dyes known asBODIPY dyes. As described below, the R1 position frequently is used inthis invention to add a derivative “tail” that may include a number ofdifferent “designer” chemical groups, the R2 and R3 positions can beused to add zwitterionic components, and the R4 position may be used tocreate other BODIPY type dyes with different colors. However, componentscan be added to different R groups as needed.

FIG. 3 depicts the general structure of an Alexa 488® (Molecular Probes)dye modified to change the molecular properties. Any of the R groups maybe used to add nontitratable charged groups to balance out the chargeson the dye to produce a zwitterionic charge balance, to add groups toreplace the titration properties of the targets of the linkers onproteins, or to add “tails” for attachment of other components that mayinclude cleavable groups and isotopic labeling groups to enhance theproperties of the optical label. The reactive tail group shown mayattach at the positions shown or may attach at one of the other Rpositions.

FIGS. 4A and 4B depicts the general structure of zwitterionic opticallabeling molecules wherein the dye group is a BODIPY dye. In FIG. 4 a,A=Ester activator, NHCH2CH2SH, or other linker; R¹ to R⁹ to be defined;R^(1, 1 to p, 1 to m) and R^(2, 1 to p, 1 to m)=to be defined; Ar=Aryl;r, n, m, p, q=0, 1, 2, 3 . . . For each value of p, there are p valuesof m. These p values can be equal or different. In FIG. 4B, A=Esteractivator, NHCH2CH2SH, or other linker; CG=Cleavable group; R¹ to R⁹=tobe defined; R^(1, 1 to p, 1 to m) and R^(2, 1 to p, 1 to m)=to bedefined; Ar=Aryl; r, n, m, p, q=0, 1, 2, 3 . . . . For each value of p,there are p values of m. These p values can be equal or different. Thedye depicted in FIG. 4B contains a cleavable group so that afterseparation of the dye-labeled proteins, the dyes can be removed toenhance enzymatic digestion of the target proteins and to simplify massspectral or other analysis of the target proteins.

FIG. 5 depicts the general structure of a zwitterionic optical labelingmolecule wherein the dye group is a BODIPY dye with a p-nitro anisolephoto-cleavable group. A=Ester activator, NHCH2CH2SH, or other linker;R¹ to R⁹=to be defined; R^(1, 1 to p, 1 to m) andR^(2, 1 to p, 1 to m)=to be defined; Ar=Aryl; r, n, m, p, q=0, 1, 2, 3 .. . . For each value of p, there are p values of m. These p values canbe equal or different.

FIGS. 6A and 6B depicts two examples of a zwitterionic optical labelingmolecule wherein the dye group is Cascade Blue dye. In FIG. 6A, n, m=1,2, 3 . . . ; R^(1 to n, 1 to m) and R^(2, 1 to n, 1 to m)=to be defined;A=nucleophilic attack activator. For each value of n, there are n valuesof m. These n values can be equal or different. In FIG. 6B, n, m=1, 2, 3. . . ; R^(1 to n, 1 to m) and R^(2, 1 to n 1 to m)=to be defined;CG=cleavable group; A=nucleophilic attack activator. For each value ofp, there are p values of m. These p values can be equal or different.The dye depicted in FIG. 6B contains a cleavable group so that afterseparation of the dye-labeled proteins, the dyes can be removed toenhance enzymatic digestions and to simplify mass spectral or otheranalysis.

FIGS. 7A and 7B depict two examples of a zwitterionic optical labelingmolecule that can be used to label phosphorylation sites on proteinsafter beta-elimination of phosphates from serine and/or threonine sidechains.

FIGS. 8A and 8B depicts the structures of zwitterionic dyes A-I.

FIGS. 9A and 9B depicts the structures of zwitterionic dyes A2-I2.

FIGS. 10A and 10B depicts the structures of zwitterionic dyes A3-I3.

FIG. 11 depicts general structures of an optical labeling moleculecomprising a zwitterionic dye moiety (ZD), a titratable group moiety (T)that closely approximates the pK of the group removed from the proteinby reaction with the functional linker, and the functional linker (A).

FIG. 12 depicts general structures of an optical labeling moleculecomprising a zwitterionic dye moiety (ZD), a titratable group moiety (T)that closely approximates the pK of the group removed from the proteinby reaction with the functional linker, a cleavable moiety (C), and thefunctional linker (A).

FIG. 13 depicts general structures of an optical labeling moleculecomprising a zwitterionic dye moiety (ZD), a titratable group moiety (T)that closely approximates the pK of the group removed from the proteinby reaction with the functional linker, a cleavable moiety (C), a secondlabel that is designed to leave a residual isotopic label on the proteinwhen the dye is removed (I), and a functional linker (A).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed toward compositions and methods usefulin the optical labeling and detection of proteins. One aspect of theinvention encompasses the use of the optical labeling molecule in thefield of proteomics. As known in the art, one of the central problemswith current proteomics methods is limited detection sensitivity. Thebest current post-labeling methods that are applied after proteinseparation (such as silver stains or fluorescent dyes) can detect lownanogram levels of protein per gel spot (Rabilloud, T., (2000) Detectingproteins separated by 2-D gel electrophoresis, Anal. Chem. 72: 48A-55A.;Berggren, K., et al., (2000) Background-free, high sensitivity stainingof proteins in one- and two-dimensional sodium dodecylsulfate-polyacrylamide gels using a luminescent ruthenium complex,Electrophoresis 21, 2509-2521), even with sophisticated laser scanners(McNamara P., et al., (2000) Fluorescent gel imaging with Typhoon 8600:Life Science News). This corresponds to detecting proteins in the rangeof about 300-3000 copies per cell under typical experimental conditions(Corthals, G. L., et al., (2000) The dynamic range of proteinexpression: a challenge for proteomic research, Electrophoresis 21:1104-1115; Patton, W. F. (2000) A thousand points of light: theapplication of fluorescence detection technologies to two-dimensionalgel electrophoresis and proteomics, Electrophoresis 21: 1123-1144),which falls short of the sensitivity needed to detect low abundanceproteins such as regulatory proteins, that are often present in low copynumber (Corthals, G. L., et al., (2000), Electrophoresis 21, 1104-1115;Gygi, S. P., et al., (2000) Evaluation of two-dimensional gelelectrophoresis-based proteome analysis technology, Proc. Natl. Acad.Sci. U.S.A 97: 9390-9395; Harry, J. L., et al., (2000) Proteomics:capacity versus utility, Electrophoresis 21: 1071-1081). Pre-labelingproteins with fluorescent dyes can maximize the sensitivity by reducingthe dye background after separation and by allowing the attachment ofone or more dyes per protein. Currently available dyes, however, sufferfrom several shortcomings. For example, the available dyes typicallyadversely affect the solubility of the proteins to which they areattached. For example, a prior report, using prelabeling withfluorescent cyanine-based dyes (Cy) and multiplex detection (Unlu, M.,et al., (1997) Difference gel electrophoresis: a single gel method fordetecting changes in protein extracts, Electrophoresis 18: 2071-2077)required a very low multiplicity of dye labeling (0.01-0.02dyes/protein) to minimize dye-induced reduction in protein solubilityand dye-induced mobility shifts, and this severely limited thesensitivity attainable.

Accordingly, the present invention provides for optical labelingmolecules that have enhanced properties for increased aqueous solubilityover a wide pH range and enhanced detection sensitivity. Preferredoptical labeling molecules of the invention are designed to containzwitterionic groups which are designed to maintain their charges over awide pH range to increase the solubility of proteins labeled with theoptical labeling molecules in both aqueous and mixed polar solventswhile minimizing pl shifts due to labeling, thereby facilitatingseparation and identification of the labeled proteins. In a preferredembodiment, the optical labeling molecule comprises a zwitterionic dyemoiety, a titratable group moiety to replace the acid-base behavior ofthe target group on proteins used for linkage and a functional linker.In a further preferred embodiment, there is more than one zwitterionicgroup present on the zwitterionic dye moiety to further enhance thesolubility of the zwitterionic dyes and the zwitterionic dye-labeledproteins over a wide pH range. The present invention in additionprovides for many channels of multiplex protein detection in a singleexperiment, by using a family of detection dyes to label proteins fromdifferent biological treatments and thus overcomes problems withexperimental reproducibility of the separations of the myriad ofproteins present in cells, organelles and in tissues.

By “optical labeling molecule” is meant any molecule useful incovalently labeling biological molecules that permits the labeledmolecule to be detected using methods that detect emission of an opticalsignal. Optical signals include, but are not limited to color,absorbance, luminescence, fluorescence, phosphorescence, withfluorescence usually being preferred for maximum detection sensitivity.That portion of the optical labeling molecule responsible for emissionof the detectable signal is referred to as the chromophore of the dyemoiety.

In a preferred embodiment of the invention, the optical labelingmolecule is detected through measuring fluorescent emission. Fluorescentemission is luminescence that is caused by the absorption of radiationat one wavelength or a band of wavelengths in its absorption band(referred to as the excitation wavelength) followed by nearly immediatereradiation, largely at a different wavelength (referred to as theemission wavelength or the emission band).

In a preferred embodiment, the optical labeling moiety comprises afluorescent dye. Suitable fluorophores include but are not limited to,fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,erythrosin, coumarin, methyl-coumarins, quantum dots (also referred toas “nanocrystals”), pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue®, Texas Red, Cy dyes (Cy2, Cy3, Cy5, Cy5.5, Cy7, etc.),alexa dyes (including, but not limited to, Alexa Fluor 350, Alexa Fluor405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750,see Molecular Probes catalog, 9th Edition), phycoerythin, BODIPY dyesand derivatives, and others described in the 9th Edition of theMolecular Probes Handbook by Richard P. Haugland, hereby expresslyincorporated by reference in its entirety. See also U.S. Pat. Nos.6,130,101, 6,162,931, 6,291,203, all of which are hereby expresslyincorporated by reference in their entirety, which depict suitable dyemoieties. The figures depict a number of suitable dye moieties for usein the invention. Additionally, it is to be understood that theinvention can be adapted by one of skill in the art to incorporateadditional existing dye chromophores or new dye chromophores

A variety of preferred dyes are depicted in the figures.

In a preferred embodiment, the optical labeling molecule comprises azwitterionic dye moiety, a titratable group moiety and a functionallinker. Zwitterionic groups are those that contain both positive andnegative charges and are net neutral, but highly charged. By“zwitterionic dye moiety” is meant a dye that is designed to contain oneor more zwitterionic groups, generally added as “zwitterioniccomponents”, e.g. separate positive and negative charged groups. Thepreferred zwitterionic dye moiety is non-titratable and thus maintainsits zwitterionic charge character over a wide pH range (e.g. 3-12), withfrom pH 4-10 and pH 5-9 and pH 6-11 being useful as well.

In a preferred embodiment, the dye moiety, preferably a fluorophore, isderivatized to include side chain groups and/or a “tail” for theaddition of components of zwitterionic charge pairs. As is shown in theFigures, any number of dyes can be derivatized to allow the additionboth of components to produce a zwitterionic charge balance and theother components appropriate for the application (e.g. titratablegroups, isotopes, linkers, etc.) of the optical labeling molecules ofthe invention.

In a preferred embodiment, the fluorophore is derivatized with an alkylor polypeptide moiety that serves as a “tail” which include componentsof zwitterionic charge pairs and a functional group for the attachmentof the other components of the labeling molecule. Preferred embodimentsinclude alkyl chains, including substituted heteroalkyl chains, andalkylaryl groups, including alkyl groups interrupted with aryl groups,or a polypeptide chain framework, as are generally depicted in thefigures.

As depicted in the figures, many of the positions of the fluorophorescan be substituted with substituent chemical groups, generally termed“R” groups herein, for a variety of purposes, as outlined herein.

In a preferred embodiment, as will be appreciated by those in the art, awide variety of possible R substituent groups may be used. Suitable Rsubstitution groups, for the structures of the invention, include, butare not limited to, hydrogen, alkyl groups including substituted alkylgroups and heteroalkyl groups as defined below, aryl groups includingsubstituted aryl and heteroaryl groups as defined below, sulfurmoieties, amine groups, oxo groups, carbonyl groups, halogens, nitrogroups, imino groups, alcohol groups, alkyoxy groups, amido groups,phosphorus moieties, ethylene glycols, ketones, aldehydes, esters,ethers, etc.

In addition, R groups on adjacent carbons, or adjacent R groups, can beattached to form cycloalkyl or cycloaryl groups, includingheterocycloalkyl and heterocycloaryl groups together with the carbonatoms of the dye. These ring structures may be similarly substituted atany position with R groups.

In addition, as will be appreciated by those skilled in the art, eachposition designated above may have two R groups attached (R′ and R″),depending on the valency of the position, although in a preferredembodiment only a single non-hydrogen R group is attached at anyparticular position; that is, preferably at least one of the R groups ateach position is hydrogen. Thus, if R is an alkyl or aryl group, thereis generally an additional hydrogen attached to the carbon, although notdepicted herein.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above. Apeptide backbone can also be used to construct the “tail” moiety whichincludes zwitterionic charge balancing components and the othercomponents of the labeling molecule.

A preferred heteroalkyl group is an alkyl amine. By “alkyl amine” orgrammatical equivalents herein is meant an alkyl group as defined above,substituted with an amine group at any position. In addition, the alkylamine may have other substitution groups, as outlined above for alkylgroup. The amine may be primary (—NH₂R), secondary (—NHRR′), or tertiary(—NRR′R″). When the amine is a secondary or tertiary amine, preferred Rgroups are alkyl groups as defined above. A preferred alkyl amine isp-aminobenzyl. When the alkyl amine serves as the coordination sitebarrier, as described below, preferred embodiments utilize the nitrogenatom of the amine as a coordination atom, for example when the alkylamine includes a pyridine or pyrrole ring.

By “aryl group” or “aromatic group” or grammatical equivalents herein ismeant an aromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc. As for alkyl groups, the arylgroup may be substituted with a substitution group, generally depictedherein as R.

By “amino groups” or grammatical equivalents herein is meant —NH₂ (aminegroups), —NHR and —NR₂ groups, with R being as defined herein. Preferredamines are tertiary (R—NR′R″) or quaternary (R—NR′R″R′″+). Preferred Rgroups are alkyl groups as defined herein. By “nitro group” herein ismeant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds (including sulfoxides (—SO—), sulfones (—SO₂₋—),sulfonates (—SO₃ ⁻), sulfates (—OSO₃ ⁻), sulfides (RSR)), thiols (—SH),and disulfides (RSSR)). By “phosphorus containing moieties” herein ismeant compounds containing phosphorus, including, but not limited to,phosphines, phosphites and phosphates. A preferred phosphorous moiety isthe —PO(OH)(R)₂ group. The phosphorus may be an alkyl phosphorus; forexample, DOTEP utilizes ethylphosphorus as a substitution group on DOTA.A preferred embodiment has a —PO(OH)₂R₂₅ group, with R₂₅ being asubstitution group as outlined herein.

By “silicon containing moieties” herein is meant compounds containingsilicon.

By “ketone” herein is meant an —RCOR— group.

By “aldehyde” herein is meant an —RCOH group.

By “ether” herein is meant an —R—O—R group.

By “alkyoxy group” herein is meant an —OR group.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(r)— group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, i.e.—(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

The optical labeling molecules, or other molecules defined herein, maycontain one or more chiral centers and/or double bonds and therefore mayexist as stereoisomers, such as double-bond isomers (i.e., geometricisomers), rotamers, enantiomers or diastereomers. Accordingly, whenstereochemistry at chiral centers is not specified, the chemicalstructures depicted herein encompass all possible configurations atthose chiral centers including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures. Enantiomeric andstereoisomeric mixtures can be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. In general, as is depictedin the figures, charged groups are added to the zwitterionic dye moiety.In general, pairs of positive and negative charged moieties (“thezwitterionic components”) are added at separate locations to the dyemoiety (see for example FIG. 1A), although in some embodiments, both thepositive and negative charges are added as single “branched” moieties(see FIG. 1B), or combinations thereof (see FIG. 1C). In someembodiments the chromophoric framework of the dye includes positively ornegatively charged groups or includes some combination of positive andnegative charges and suitable charge groups added to make the number ofpositive and negative groups equal (in order to form zwitterionicpairs). In some embodiments, the actual fluorophore has a derivative“tail”, used as a linker to the other components of the optical labelingmoiety, which can contain zwitterionic components as well (see FIGS. 1Dand 1E). It should be noted that for purposes of the invention, thesederivatives are included in the definition of “dye moiety”. Inadditional embodiments, the zwitterionic components are added anywherewithin the optical labeling moiety; for example, negative charges can beadded to the fluorophore, and positive charges to the linker moiety, orvice versa.

Particularly preferred zwitterionic components are small alkyl groups(C2-C3) with quaternary ammonium groups (—NR3+), guanidine groups, orother positively charged groups which are not titratable until the edgeof the most basic regions of interest, and negatively charged alkylsulfonate or alkyl sulfate groups. Any other charged groups that are nottitratable between pH 3-12 and are stable under aqueous conditions aresuitable to include as components of zwitterionic groups.

In a further preferred embodiment, the zwitterionic substitution of one,two or more quaternary ammonium group and one, two or more sulfonategroups are added to one of the family of boron difluoridediaza-indacene-propionic acid (BODIPY) dyes. The BODIPY family of dyesare stable molecules that have dyes have many favorable properties foruse as the neutral dye moiety (Johnson, I. D., et al., (1991),Fluorescent membrane probes incorporating dipyrrometheneboron difluoridefluorophores, Anal. Biochem 198: 228-237; Karolin, J., et al., (1994)Fluorescence and absorption spectroscopic properties ofdipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipidmembranes, and proteins, J. Am. Chem. Soc. 116: 7801-7806, each of whichare hereby incorporated by reference). BODIPY dyes have high sensitivity(extinction coefficient >70,000 cm⁻¹M⁻¹ and quantum yield 0.5-1.0),their fluorescence signals are insensitive to solvent and pH, and theyexhibit high chemical and photo stability (Vos de Wael, E., et al.,(1977) Pyromethene-BF2 complexes(4,4″-difluoro-4-bora-3a,4a-diaza-s-indacenes), Synthesis andluminescence properties, Recl. Trav. Chim. Pays-Bas 96: 306-309;Haugland, R. P. and Kang, H. C. Chemically Reactive DipyrrometheneBoronDifluoride Dyes, Molecular Probes, Inc. 83,458[4,774,339], 1-14.1988,each of which are hereby incorporated by reference). BODIPY dyes havenarrow excitation spectra and a wide range of excitation/emissionspectra are available in the different members of the series (9thEdition of the Molecular Probes Handbook, hereby expressly incorporatedby reference), which facilitates the design and implementation of themultiplex protein detection techniques of this invention. Members of theBODIPY family of dyes have very similar structures but have differentexcitation and emission spectra that allows multiplex detection ofproteins from two or more protein sample mixtures simultaneously on thesame gel. Multiplex detection, or multiplexing, is defined as thetransmission of two or more messages simultaneously with subsequentseparation of the signals at the receiver. Specific examples of BODIPYdyes that have been engineered to contain one zwitterionic group areshown in FIGS. 8A and 8B as dyes A-G, in FIGS. 9A and 9B as dyes A2-G2,and in FIGS. 10A and 10B as dyes A3-G3.

In another preferred embodiment, a double zwitterionic substitution oftwo quaternary ammonium and two sulfonate groups are added to a neutraldye moiety. In a further preferred embodiment, the double zwitterionicsubstitution of two quaternary ammonium and sulfonate groups are addedto a BODIPY dye moiety. Specific examples of BODIPY dyes that have beenengineered to contain two zwitterionic groups are shown in FIG. 8B asdyes H and I, in FIG. 9B as dyes H2 and I2 and in FIG. 10B as dyes H3and I3.

In general, dyes A, C, E and H (including the dyes designated A2, A3,C2, C3, E2, E3 and H2, H3) have an excitation/emission spectra of528/547 nm and are efficiently excited by 488 or 532 nm lasers. Dyes B,D F and I (including the dyes designated B2, B3, D2, D3, F2, F3, I2 andI3) have an excitation/emission spectra of 630/650 nm and areefficiently excited by 633 nm lasers. Dye G (including the dyesdesignated G2, G3) has an excitation/emission spectra of 588/616 nm andis efficiently excited by 532 nm lasers. However, these numbers may varyslightly. Dyes from the first two groups, for example dye A and dye B,have exceedingly low optical “cross-talk” when excited at 488 or 633 nm,respectively, so that the excitation and emission of each group does notexcite the other group and the signals from the two groups are wellseparated.

The spectra of dye G fits sufficiently well between the other two groupsof dyes that three-color experiments can be done with 488, 532 and 633nm lasers combined with suitable optical filters to differentiate theemission of the dyes. Measuring full emission spectra from spots on 2Dgels will allow the effective separation of the signals from dyes thathave strongly overlapping emission spectra and allow the simultaneoususe of many similar dyes with slightly different spectra to carry outefficient multiplex detection of proteins with a much larger differentnumbers of color channels. The compounds of the invention areparticularly suited for such use.

Example 1 describes a route of synthesis for dyes A-I.

In another embodiment of the invention, the positions of quaternaryammonium and sulfonate groups of the dyes A-I are switched to form dyesA2-I2 as indicated in FIGS. 9A and 9B.

Example 2 describes a route of synthesis for dyes A2-I2.

In another embodiment of the invention, the arginine residues of dyesA2-I2 are substituted with trimethylated lysines to form dyes A3-I3 asindicated in FIGS. 10A and 10B.

Example 3 describes a route of synthesis for dyes A3-I3.

There are two general ways to make optical labeling molecules of theinvention. The first way is exemplified by Cascade blue or Alexa dyeswhere the dye structure is relatively polar and compact but there is anet charge on the dye that would substantially alter the isoelectricpoints of labeled proteins. To overcome this problem, a tail can bedesigned and added to include nontitratable opposing charges to formnontitratable zwitterionic charge pairs, to add additional zwitterioniccharge pairs, to add titratable groups to replace the acid/baseproperties of protein groups that are modified by the linker, to add anoptional cleavable group, to add an optional second label stable isotopegroup, and to add a linker group, as described above. The second way tomake zwitterionic dyes is exemplified by the BODIPY example, wherecomponents of the dye are designed, synthesized and assembled to achievethe dye properties desired. Briefly, steps of organic synthesis aredesigned to incorporate one or more nontitratable zwitterionic chargepairs, to add titratable groups to replace the acid/base properties ofprotein groups that are modified by the linker, to add an optionalcleavable group, to add an optional second label stable isotope group,and to add a linker group, as described above.

In a preferred embodiment, in addition to the zwitterionic dye moiety,the optical labeling molecule further comprises a titratable groupmoiety and a functional linker. By “titratable group moiety” is meant agroup that mimics the acid-base titration of the group labeled on thetarget molecule. The charge on the group labeled on the target moleculeis typically lost when the group labeled on the target molecule forms acovalent bond with the functional linker of the optical labelingmolecule. The titratable group moiety replaces the lost charge and thusmaintains the isoelectric points of the labeled target molecules. Asdiscussed herein, in a preferred embodiment of the invention, the targetmolecule is a protein. In this situation, the titratable group replacesthe charge lost when the functional linker forms a covalent bound withthe protein, thus closely maintaining the protein's isoelectric point.The isoelectric points of proteins are important factors in determiningseparation of the proteins using techniques based on the charge and sizecharacteristics such as two-dimensional electrophoresis, ion exchangechromatography, or capillary electrophoresis.

In a further preferred embodiment, in addition to the zwitterionic dyemoiety and the titratable group moiety, the optical labeling moleculefurther comprises a functional linker. This linker is used to attach theoptical labeling molecule to the target molecule. Linkers are well knownin the art; for example, homo- or hetero-bifunctional linkers are wellknown (see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, hereby expressly incorporated byreference). Preferred linkers include, but are not limited to,succinimidyl groups, sulfosuccinimidyl groups, imido esters,isothiocyanates, aldehydes, sulfonylchlorides, arylating agents,maleimides, iodoacetamides, alkyl bromides, or benzoxidiazoles.

The linker forms a covalent bond with one or more sites on a targetprotein. As will be appreciated by those in the art, there are a largenumber of possible proteinaceous target analytes that may be detectedusing the present invention. By “proteins” or grammatical equivalentsherein is meant proteins, oligopeptides and peptides, derivatives andanalogs, including proteins containing non-naturally occurring aminoacids and amino acid analogs, and peptidomimetic structures. The sidechains may be in either the (R) or the (S) configuration. In a preferredembodiment, the amino acids are in the (S) or L-configuration.

In a preferred embodiment, the type and number of proteins to be labeledwill be determined by the method or desired result. In some instances,most or all of the proteins of a cell or virus are labeled; in otherinstances, some subset, for example subcellular fractionation, is firstcarried out, or macromolecular protein complexes are first isolated, asis known in the art, before dye labeling, protein separation andanalysis.

Target proteins of the invention include all cellular proteins.Preferred target proteins include regulatory proteins such as receptorsand transcription factors as well as structural proteins.

Further preferred target proteins include enzymes. As will beappreciated by those in the art, any number of different enzymes can belabeled. The enzymes (or other proteins) may be from any organisms,including prokaryotes and eukaryotes, with enzymes from bacteria, fungi,extremeophiles, viruses, animals (particularly mammals and particularlyhuman) and birds all possible. Suitable classes of enzymes include, butare not limited to, hydrolases such as proteases, carbohydrases,lipases; isomerases such as racemases, epimerases, tautomerases, ormutases; transferases, kinases and phosphatases. Preferred enzymesinclude those that carry out group transfers, such as acyl grouptransfers, including endo- and exopeptidases (serine, cysteine, metalloand acid proteases); amino group and glutamyl transfers, includingglutaminases, γ glutamyl transpeptidases, amidotransferases, etc.;phosphoryl group transfers, including phosphotases, phosphodiesterases,kinases, and phosphorylases; nucleotidyl and pyrophosphotyl transfers,including carboxylate, pyrophosphoryl transfers, etc.; glycosyl grouptransfers; enzymes that do enzymatic oxidation and reduction, such asdehydrogenases, monooxygenases, oxidases, hydroxylases, reductases,etc.; enzymes that catalyze eliminations, isomerizations andrearrangements, such as elimination/addition of water using aconitase,fumarase, enolase, crotonase, carbon-nitrogen lyases, etc.; and enzymesthat make or break carbon-carbon bonds, i.e. carbanion reactions.Suitable enzymes are listed in the Swiss-Prot enzyme database.

Suitable viruses as sources of analytes to be labeled include, but arenot limited to, orthomyxoviruses, (e.g. influenza virus),paramyxoviruses (e.g. respiratory syncytial virus, mumps virus, measlesvirus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella□zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and-II), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like) Suitable bacteria include, but are notlimited to, Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g.Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g.S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium,e.g. C. botulinum, C. tetani, C. difficile, C. perfringens;Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S.pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H.influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia,e.g. G. lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like.

In addition, any number of different cell types or cell lines may beevaluated using the labeling molecules of the invention.

Particularly preferred are disease state cell types, including, but arenot limited to, tumor cells of all types (particularly melanoma, myeloidleukemia, carcinomas of the lung, breast, ovaries, colon, kidney,prostate, pancreas and testes), cardiomyocytes, endothelial cells,epithelial cells, lymphocytes (T-cell and B cell), mast cells,eosinophils, vascular intimal cells, hepatocytes, leukocytes includingmononuclear leukocytes, stem cells such as haemopoetic, neural, skin,lung, kidney, liver and myocyte stem cells (for use in screening fordifferentiation and de-differentiation factors), osteoclasts,chondrocytes and other connective tissue cells, keratinocytes,melanocytes, liver cells, kidney cells, and adipocytes. Suitable cellsalso include known research cell lines, including, but not limited to,Jurkat T cells, NIH3T3 cells, CHO, Cos, etc. See the ATCC cell linecatalog, hereby expressly incorporated by reference.

In one embodiment, the cells may be genetically engineered, that is,contain exogeneous nucleic acid, for example, when the effect ofadditional genes or regulatory sequences on expressed proteins is to beevaluated.

In some embodiments, the target analyte may not be a protein; that is,in some instances, as will be appreciated by those in the art, othercellular components, including carbohydrates, lipids, nucleic acids,etc., can be labeled as well. In general this is done using the same orsimilar types of chemistry except that the linker moieties may bedifferent and there may or may not be a need for a titratable group inthe dye to maintain the pl of the labeled molecule, as will beappreciated by those in the art.

As will be appreciated by those in the art, depending on the targetmolecule(s), an appropriate linker is chosen.

In a preferred embodiment of the invention, the linker forms a covalentbond with an amine group of the target protein. Examples of linkers thatform covalent bonds with amine groups are imidoesters andN-hydroxysuccinimidyl esters, sulfosuccinimidyl esters, isothiocyanates,aldehydes, sulfonylchlorides, or arylating agents. Amine groups arepresent in several amino acids, including lysine. Lysine ε-amino groupsare very common in proteins (typically 6-7/100 of the residues) and thevast majority of the lysines are located on protein surfaces, wheretypically they are accessible to labeling. In a preferred embodiment ofthe invention, the more reactive N-terminal amino groups may bepre-labeled near neutral pH with a different amine-reactive group, suchas a small acid anhydride with or without an isotopic label to minimizedye-induced shifts in isoelectric focusing after lysine labeling. Smallisotope-labeled groups on the N-terminus can be used for independentprotein quantitation, using isotope ratio measurements in a massspectrometer. The surface-exposed lysine amino groups tend to have pKsvery close to 10 (Tanford, C. (1962) The interpretation of hydrogen iontitration curves of proteins. Adv. Protein Chem. 17: 69-165; Mattew, J.B., et al., (1985) pH-dependent processes in proteins, CRC Crit. Rev.Biochem 18: 91-197, each of which are hereby expressly incorporated byreference) react at higher pH and their pKs can be mimicked by(hindered, non-reactive) amino groups added as the titratable groupmoiety in the optical labeling molecules of the invention.

In another embodiment of the invention, thiol groups of the targetprotein are used as the linker attachment site. Examples of linkers thatform covalent bonds with thiol groups are sulfhydryl-reactivemaleimides, iodoacetamides, alkyl bromides, or benzoxidiazoles.

The covalent bond is formed between the functional linker and targetprotein under conditions well known in the art and further discussedherein.

Thus, in a preferred embodiment of the invention, the optical labelingmolecule has one or more zwitterionic dye moiety, a titratable groupmoiety, and a functional linker and has one of the general structuresdepicted in FIG. 11.

In a preferred embodiment, in addition to the zwitterionic dye moiety,the titratable group moiety, and the functional linker, the opticallabeling molecule further comprises a cleavable moiety. By “cleavablemoiety” is meant a group that can be chemically, photochemically, orenzymatically cleaved. In a preferred embodiment of the invention, thecleavable moiety is a moiety that forms a stable bond but can beefficiently cleaved under mild, preferably physiological, conditions. Ina preferred embodiment, the cleavage site utilizes a photocleavablemoiety. That is, upon exposure to suitable wavelengths of light absorbedby the photo-cleavable groups, cleavage of the linker occurs, therebyremoving the dye from the protein or other molecule to facilitatefurther analysis. A particularly preferred class of photocleavablemoieties are the O-nitrobenzylic compounds, which can be syntheticallyincorporated into the zwitterionic labeling dye via an ether, thioether,ester (including phosphate esters), amine or similar linkage to aheteroatom (particularly oxygen, nitrogen or sulfur). Also of use arebenzoin-based photocleavable moieties. Nitrophenylcarbamate esters areparticularly preferred. A wide variety of suitable photocleavablemoieties is outlined in the Molecular Probes Catalog, supra.

By engineering in a cleavable moiety on the optical labeling molecule,the maximum detection sensitivity of the labeling molecule is increasedby allowing a high multiplicity of dye labeling that will increase themaximum detection sensitivity, followed by removal of the labelingmolecule prior to further analysis. For example, the optical labelingmolecule can be removed after protein separation via cleavage of thecleavable moiety prior to mass spectroscopy (MS) analysis.Identification of interesting protein spots on 2D gels for further studyis typically accomplished by fluorescent scanning during analysis of thegels, but identification of the proteins contained in those spots isgenerally accomplished by mass spectrometry. The most generallyeffective method of identifying proteins and post-translationalmodifications digests proteins with trypsin or other lysine-specificenzymes, before analysis by mass spectrometry. As is well known in theart, trypsin is an enzyme that specifically cleaves at the basic aminoacid groups, arginine and lysine. High multiplicity attachment ofoptical labeling molecules on amino groups will “cover” some of the mostaccessible lysine amino groups and if the dyes are not removed they willinhibit trypsin digestion at these sites. In some embodiments, this maybe preferred In some embodiments, this may be preferred. Thus, theremoval of the dye after protein separation by chemical, photochemicalor enzymatic cleavage is preferable in some embodiments.

In a further embodiment of the invention, the optical labeling moleculehas a zwitterionic dye moiety, a titratable group moiety, a functionallinker, and a cleavable moiety and has one of the general structures asdepicted in FIG. 12.

In a further embodiment of the invention, the optical labeling moleculecomprises a second label in addition to the zwitterionic dye. A secondlabel can, for example, be a stable isotope label, an affinity tag, anenzymatic label, a magnetic label, or a second fluorophore.

In a preferred embodiment of the invention, the optical labeling moietycomprises a zwitterionic dye moiety, a titratable group moiety, acleavable moiety, a stable isotope moiety, and a functional linker. In apreferred embodiment of the invention, the stable isotope moiety made upof light isotopes. In a further preferred embodiment, the stable isotopemoiety is one or more combinations of heavy isotopes. In one embodimentof the invention, the stable isotope is located between the cleavablemoiety and the functional linker.

Thus, in a preferred embodiment of the invention, the optical labelingmolecule has a zwitterionic dye moiety, a titratable group moiety, acleavable moiety, a stable isotope moiety, and a functional linker andhas one of the general structure as depicted in FIG. 13. With thisembodiment, when the cleavable moiety is cleaved, the stable isotopemoiety is left on the protein and the relative amount of the proteinexpressed by the biological system under different stimulus conditionscan be quantitated using isotope ratios in a mass spectrometer.

Another embodiment of the invention is a target molecule labeled with anoptical labeling molecule as described in any of the previouslydiscussed embodiments.

Once made, the compositions of the invention find use in a wide varietyof applications.

One aspect of the invention provides for a method of labeling a proteinusing any of the above-described optical labeling molecules wherein theoptical labeling molecule is contacted with a target protein to form alabeled protein. The event of contacting the target protein with anoptical label of the invention is also referred to as a labelingreaction. As is known in the art, conditions that may affect theefficiency of the labeling reaction include the sensitivity of labelingreaction to pH, buffer type, and the salts in the reaction medium. Inone embodiment of the invention, the labeling reaction is performed nearpH 8.5. Amine-containing buffers are generally avoided to preventpotential cross-reactions with the amine reactive functional linkergroups when such groups are used. Preferred buffers include, but are notlimited to, phosphate, phosphate/borate, tertiary amine buffers such asBICINE, and borate. Additional agents that may be added to the labelingreaction included various detergents, urea, and thiourea.

The efficiency and progress of the labeling reaction, also referred toas labeling kinetics, and can be measured by quenching the labelingreaction at different times with excess glycine, hydroxylamine or otheramine. The number of dyes per labeled protein and the relativefluorescence of the dyes on different labeled proteins can be determinedusing methods well known to those of skill in the art. For example, thenumber of optical labeling molecules per labeled protein and therelative fluorescence of the optical labeling molecules on differentlabeled proteins can be determined by separating the labeled proteinsfrom the free optical label, using HPLC gel filtration with in-linefluorescence and absorbance detection. The ratio of hydrolyzed andunreacted optical label can be determined on the free optical labelfraction by RP-HPLC (reverse-phase HPLC), if desired to help optimizelabeling conditions. Isolated, labeled proteins can be incubated and runagain on gel filtration determine the stability of protein-optical labelmolecule. (Miyairi S., et al., (1998) Determination of metallothioneinby high-performance liquid chromatography with fluorescence detectionusing an isocratic solvent system. Anal Biochem. 258(2):168-75; Mills JS, et al. (1998), Identification of a ligand binding site in the humanneutrophil formyl peptide receptor using a site-specific fluorescentphotoaffinity label and mass spectrometry, J Biol. Chem.273(17):10428-35; Kwon G, et al., (1993) Synthesis and characterizationof fluorescently labeled bovine brain G protein subunits, Biochemistry,32(9):2401-8, each of which is hereby expressly incorporated byreference).

In a further embodiment of the invention, a plurality of target proteinsare labeled with different optical labeling molecules of the invention.By “different optical labeling molecule” is meant optical labelingmolecules of the invention that are preferably but not necessarily fromthe same family, but exhibit different optical properties. For example,one family of different optical labeling molecules is a number ofoptical labeling molecules with fluorescent zwitterionic dye moieties,where each one of the family exhibits a different fluorescence spectra.Preferably, but not required, each optical labeling molecule of thefamily has similar physical characteristics. By “similar physicalcharacteristics” is meant that each optical labeling molecule of thefamily has similar size charge and isoelectric point characteristics tominimize any shifts in isoelectric point or ion exchange chromatographicmobility between the labeled and unlabeled proteins. Optical labelingmolecules that have similar physical characteristics are preferable tominimize any relative changes in physical characteristics of the proteinthat arise as a result of the presence of the optical labeling moleculeon the protein. For example, the presence of a labeling molecule on theprotein may result in a change in the gel mobility or electrophoresismobility of the labeled protein relative to the unlabeled protein. Ifeach labeling molecule of the family has similar physicalcharacteristics, the plurality of labeled proteins labeled withdifferent dyes will retain sufficiently similar physical characteristicsto minimize differences in separation.

One of the most sensitive protein parameters in 2D gel analysis that canbe perturbed by dye labeling is the isoelectric point and solubility ofthe labeled molecule at or near the isoelectric point. 2D gels havemodest resolution by mass and so labeling with different numbers of dyesgenerally does not change the apparent mass in a significant manner on2D gels. The nontitratable zwitterionic dyes of the invention increasethe solubility of proteins especially at the isoelectric point but donot change the isoelectric point of the protein significantly andtitratable groups that replace the acid/base behavior of the target ofthe dye linker group on the protein minimize isoelectric point shifts inthe labeled protein. As a result, the plurality of proteins labeled withdifferent dyes generally exhibit virtually the same gel mobility orelectrophoresis mobility pattern and will also be very similar to theunlabeled proteins.

In a preferred embodiment of the invention, the family of differentoptical labeling molecules is selected from dyes A-I (FIGS. 8A and 8B).In another preferred embodiment of the invention, the family ofdifferent optical labeling molecules is selected from dyes A2-I2 (FIGS.9A and 9B). In yet another preferred embodiment of the invention, thefamily of different optical labeling molecules is selected from dyesA3-I3 (FIGS. 10A and 10B).

The invention finds utility in a number of applications including use infield of proteomics. The optical labeling molecules of the invention canbe used to identify “functional proteomes”—namely cellular proteins thatchange in level of expression and/or post-translational modification inresponse to physiological stimuli.

It is an aspect of the invention to provide optical labeling moleculeswith improved properties for use in multiplex detection reactions ofproteins in proteomics. Thus, the invention provides for a family ofdifferent optical labeling molecules for use in labeling a plurality oftarget proteins. As discussed above, each member of the zwitterionic dyelabeling reagent family exhibits different optical properties, however,each optical labeling molecule of a dye family has quite similarphysical characteristics to other optical labeling molecules of the samefamily.

In general, a proteomics experiment typically involves the analysis ofthe proteins present in a cellular extract of the intact organism,tissue, cell or subcellular fraction before and after exposure to aparticular physiological stimulus. In one embodiment, proteins that arepresent in the extract of the cells prior to exposure to thephysiological stimuli are labeled with one of the optical labelingmolecules. Proteins that are present in the extract of the cells afterexposure to the physiological stimuli are labeled with a different oneof the optical labeling molecule family, after different strengths ofphysiological stimuli are applied. Additional samples may be labeledwith additional different optical labeling molecules. The dye labeledproteins from two or more cellular extracts are mixed and thensimultaneously separated and analyzed by observing the optical signalsof the separated proteins, thus permitting the identification of theproteins which are detectably altered in expression level orpost-translational modification state in response to the stimuli ofinterest and facilitating a further focused study of these proteins andtheir post-translational modifications. In one embodiment of theinvention, the presence or absence of the labeled proteins is analyzedto determine if a specific protein is affected by the presence orabsence of the physiological stimuli. In a further embodiment of theinvention, the relative quantity (or ratios of expression) of thespecific labeled proteins is determined.

In a preferred embodiment, the plurality of different labeled proteinsare separated prior to determining the ratios of expression orpost-translational modification of the different labeled proteins. Thedifferent labeled proteins may be separated using, for example, 1D gelelectrophoresis, 2D gel electrophoresis, capillary electrophoresis, 1Dchromatography, 2D chromatography, 3D chromatography, or massspectroscopy. In a preferred embodiment of the invention, the largenumber of labeled proteins are separated by 2D gel electrophoresis andthe relative amounts of the proteins in different spots are determinedby laser densitometry and multiplex analysis of the strength of thefluorescence of the different dye signals.

The effect of dye labeling on protein solubility and mobility duringseparation techniques, including two-dimensional electrophoresisanalysis, can also be assessed using methods known in the art. Forexample, the solubility of labeled proteins can be measured by firstradioactive N-acetyl labeling, largely of N-terminal groups nearneutrality, followed by fluorescent dye labeling of the epsilon aminogroups of lysine at elevated pH. An alternative method of radioactivelabeling will reduce sulfhydryl groups with tributyl phosphine (TBP)and/or tricarboxyethyl phosphine (TCEP) or tri-(2 cyano ethyl)phosphineand label the sulfhydryl groups with radioactive iodoacetaarude,followed by amino group dye labeling. Next, 2D gels can be run on theradioactively tagged and labeled proteins after low (substoichiometric),medium (one or two optical labeling molecules per protein) and highlabeling (many optical labeling molecules per protein). The gels canthen be scanned for fluorescence and the location of radioactive spotscan be measured by phosphorimaging on the same instrument, for examplethe BioRadFX Fluorescent Gel Scanner and Phosphoimager. The solubilitiesof labeled proteins can be assessed from changes of retention ofproteins on the IEF strips and band streaking in the second dimension,which occurs with insufficient solubility.

Any labeling molecule-induced shifts in protein patterns can bemonitored and the expected reduction of shifts assessed using the dyeswith titratable groups. The labeling conditions can be optimized formaximum sensitivity with minimum acceptable mobility shifts.

In yet a further aspect of the invention, the different labeled proteinsare further analyzed to determine the relative quantity of eachdifferent labeled protein. The relative quantity of the differentlabeled proteins can be determined, for example, by measuring therelative intensity of the optical signal emitted by each of thedifferent labeled proteins.

In a further aspect of the invention, the different labeled proteins arefurther analyzed to determine absolute quantity. Absolute quantity of alabeled protein can be determined, for example, by including a knownamount of an optically labeled protein as an internal standard. Absolutequantity can also be determined by including a known amount of anisotopically-labeled protein or peptide as an internal standard.

In yet a further aspect of the invention, a cleavable group moiety ispresent on the optical labeling molecule between the zwitterionic dyemoiety and the functional linker moiety. After separating the differentlabeled proteins as discussed above, the cleavable moiety is cleaved toremove the optical labeling molecule from the target protein. The targetprotein can then be analyzed, for example, using mass spectraltechniques (Tao, W. A. and Aebersold, R., (2003) Advances inquantitative proteomics via stable isotope tagging and massspectrometry, Current Opinion in Biotechnology, 14:110-188; Yates, J. R.III (2000) Mass spectrometry. From genomics to proteomics, Trends Genet.16: 5-8, each of which is hereby expressly incorporated by reference).

In a further aspect of the invention, the various post-translationalmodifications are identified. Post-translational modifications includephosphorylation, methionine oxidation, cysteine oxidation to sulfenicacid, tyrosine nitration, thiol nitrosylation, disulfide formation,glycoslyation, carboxylation, acylation, methylation, sulfation, andprenylation.

In a preferred embodiment of the invention, the phosphorylation state ofthe proteins in the cells is determined. In this embodiment,unstimulated cells are labeled with ³³P phosphate and the proteinextract of the cells labeled with a first optical labeling molecule.Cells that have been exposed to a growth factor or other stimulus arelabeled with ³²P phosphate and a second different optical labelingmolecule. Preferably, the first and the second optical labelingmolecules are chosen from the same set of optical labeling molecules sothat the optical signal is different but the physical characteristicsare similar. The labeled extracts of the cells are mixed andsimultaneously separated by a method described above. The labeledextracts are analyzed with optical scanning to determine proteinexpression ratios between the stimulated and unstimulated cells. The gelis sandwiched between two phosphoimaging detector plates with a thinmetal foil in between the gel and the phosphoimager plate on one side ofthe gel. The phosphoimager plate on the side with no foil responds to³²P+³³P whereas the phosphoimager plate on the side with the metal foilonly detects the ³²P since the beta radiation from the ³³P is blocked bythe thin metal foil. The phosphoimager plates are read and the ratios ofthe signals for the two plates are analyzed to determine the relativeamount of phosphorylation on each protein on the gel. The methods can beused to determine the levels of phosphorylation of each protein on a gelby using antibodies or other labels, e.g. antiphosphothreonineantibodies (that are well known) and a chemical labeling method forphosphoserine and phosphothreonine groups on gel-separated proteins.After the proteins are separated on the gel and expression ratiosmeasured by laser scanning the gels, the proteins can either be furtheranalyzed on the gel or transferred to blotting membranes for furtheranalysis.

In order to measure the phosphoserine and phosphothreonine levels oneach protein, one embodiment is to incubate the gel or blot in strongbase (e.g. 1 M barium hydroxide) at 60 degrees C. for several hours tobeta-eliminate the phosphate groups from phosphoserine andphosphothreonine. A dye similar to or identical to one of the dyes shownin FIG. 7A or FIG. 7B is reacted with the modified proteins, the excessunreacted dye is rinsed away and fluorescence signals that reflectprotein phosphorylation are measured. Other methods are available todetect other post-translational modifications of proteins by pre- orpost-labeling on gels where protein expression ratios have beenmeasured. Thus, the protein multiplex methods of the invention can beextended for with simultaneous monitoring of changes in phosphorylation,as well as the changes in the level of the protein and otherpostranslational modifications of the proteins.

A further aspect of the invention provides for methods of determiningwhether a particular protein is exposed to the surface of its nativeenvironment. In one embodiment of the invention, a first opticallabeling molecule is used to label exposed target proteins on thesurfaces of cells, isolated organelles or isolated multiproteincomplexes. The cell or organelle membranes or the multiprotein complexstructure are then disrupted with detergents and/or chaotropic compoundsand the interior groups labeled with a second, different opticallabeling molecule. The sample is then separated by a method describedabove. Those proteins labeled with the first optical labeling moleculeare proteins exposed to the surface of the cell, organelle ormultiprotein complex. Those proteins labeled with the second opticallabeling molecule are proteins that are not exposed to the surface ofcell, organelle or multiprotein complex. In a preferred embodiment ofthe invention, the labeled proteins are isolated and identified, asdescribed above.

In addition, as will be appreciated by those in the art, thecompositions of the invention can be used as optical labels in anystandard application of optical labels. For example, the analysis ofsingle proteins can be done. A wide variety of techniques andapplications are described in the gth ed. of the Molecular ProbesCatalog and references cited therein. Similarly, certain nucleic acidanalyses such as gene expression and genotyping utilize dyes, which canbe the dyes of the invention. For example, capillary electrophoresisseparations of both proteins and nucleic acids can rely on pl, and thedyes of the invention can be used in these applications.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are hereby expressly incorporated byreference.

Additional references, each of which is hereby incorporated byreference:

-   1. Holt, L. J., et al., (2000) The use of recombinant antibodies in    proteomics. Curr. Opin. Biotechnol. 11: 445-449.-   2. Unlu, M., et al., (1997) Difference gel electrophoresis: a single    gel method for detecting changes in protein extracts,    Electrophoresis 18: 2071-2077.-   3. Griffiths, W. J. (2000) Nanospray mass spectrometry in protein    and peptide chemistry. EXS 88: 69-79.-   4. Borchers, C., et al., (2000) Identification of in-gel digested    proteins by complementary peptide mass fingerprinting and tandem    mass spectrometry data obtained on an electrospray ionization    quadrupole time-of-flight mass spectrometer, Anal. Chem. 72:    1163-1168.-   5. Belov, M. E., et al., (2000) Zeptomole-sensitivity electrospray    ionization—Fourier transform ion cyclotron resonance mass    spectrometry of proteins, Anal. Chem. 72: 2271-2279.-   6. Gatlin, C. L., et al., (1998) Protein identification at the low    femtomole level from silver-stained gels using a new fritless    electrospray interface for liquid chromatography-microspray and    nanospray mass spectrometry, Anal. Biochem. 263: 93-101.-   7. Ogueta, S., et al., (2000) Identification of phosphorylation    sites in proteins by nanospray quadrupole ion trap mass    spectrometry, J. Mass Spectrom. 35: 556-565.-   8. Loo, J. A., et al., (1999) High sensitivity mass spectrometric    methods for obtaining intact molecular weights from gel-separated    proteins, Electrophoresis 20: 743-748.-   9. Cordwell, S. J., et al., (2000) Subproteomics based upon protein    cellular location and relative solubilities in conjunction with    composite two-dimensional electrophoresis gels. Electrophoresis 21:    1094-1103.

EXAMPLE 1 Synthesis of Dyes A-I

The synthetic scheme and description below provides an example ofsynthesis for dyes A-I (FIGS. 8A and 8B). All references listed beloware hereby expressly incorporated by reference.

The synthesis of engineered dye A for proteomic analyses requiressequential coupling of the synthetic boradiazaindacene-3-propionic acid,succinimidyl ester 1, prepared as outlined in Scheme 1, with glycine andL-Cys(SO₃H)—OH which will provide acid 2. Direct coupling of I withGly-L-Cys(SO₃H)—OH leads directly to 2. Activation (Delfino, J. M., etal., (1993) Design, Synthesis, and Properties of a PhotoactivatableMembrane-Spanning Phospholipidic Probe. J. Am. Chem. Soc., 115:3458-3474) of 2 in acetonitrile with commercially availableN-hydroxysulfosuccinimide sodium salt (3) and DMAP followed by additionof DCC will generate A. The synthesis of 1 commences with the knownpyrrole 4 (Bray, B. L.; et al., (1990) J. Org. Chem., 55, 6317) and thereadily available pyrrole 11 (Muchowski, J. M. and Hess, P., “Lithiationof the dimer of 3-bromo-6-dimethylamino-1-azafulvene. Efficacioussynthesis of 4-mono- and 4,5-disubstituted pyrrole-2-carboxaldehydes.”(1988) Tetrahedron Lett., 29(26), 3215). Bromination of 4 using NBSprovided 5 which underwent Suzuki coupling with phenylboronic acid toyield 2-phenyl-4-formylpyrrole 6. Ester 7 was obtained through a Doebnercondensation of 6 with mono-ethyl malonic acid followed by catalytichydrogenation of the resulting olefin. Conversion of the esterfunctionality in 7 to the corresponding dimethylamine was carried out intwo steps. Treatment of 7 with dimethylamonium chloride in the presenceof trimethyl aluminum led to the corresponding N,N-dimethyl amide whichwas subsequently reduced into the amine by treatment with lithiumaluminum hydride (LAH), formylated under the Vilsmeier-Haack reactionconditions, and treated with methyliodide to give way to formyl pyrrole8 which upon condensation with pyrrole 11 afforded 9. Exposure of 9 toborontrifluoride etherate in the presence of Hunig's base using amodification of Lugtenburg's protocol (Vos de Wael, E., et al., (1977)Pyromethene-BF₂ complexes(4,4″-difluoro-4-bora-3a,4a-diaza-s-indacenes), Synthesis andluminescence properties. Recl. Trav. Chim. Pays-Bas 96, 306-309) gaverise to the difluoroboradiaza-indacene 10. Preparation of thesuccinimidyl ester 1 required treatment of 10 with N-hydroxysuccinimideand DCC in acetonitrile. Manipulation of the resulting carbonyl groupsis straightforward.

The synthesis of dye B requires condensation of the syntheticboradiazaindacene aldehyde 14 with the readily available ylid 13 leadingto 15. Formation of the corresponding sulfosuccinimidyl ester, followedby addition of L-Cys(SO₃ ⁻Na⁺)—OH, provides 16 which can be transformedinto the target dye B employing 3. The required aldehyde 14 is preparedfrom the readily available pyrrole 17a (Sambrotta, L., et al., (1989)Synthesis of 8-Demethyl-8-Formyl Protoporphyrin IX and of 8-DemethylProtoporphyrin IX, Tetrahedron 45: 6645-6652.) and the known pyrrole 21(Barton, D. H. R, et al., (1990) A Useful Synthesis of Pyrroles fromNitroolefins, Tetrahedron 46[21], 7587-7598, hereby expressly) asillustrated in Scheme 2.

Employing the two-step protocol of Boger (Boger, D. L., and Patel, M.(1988) Total Synthesis of Prodigiosin, Prodigiosene, andDesmethoxyprodigiosin: Diels-Alder Reactions of Heterocyclic Azadienesand Development of an Effective Palladium (II)-Promoted 2,2′-BipyrroleCoupling Procedure, J. Org. Chem., 53, 1405-1415) for the preparation of2,2′-bispyrroles, pyrrole-1-carboxylic acid is treated withtriphenylphosphine-carbon tetrachloride followed by the addition of thesodium salt (17b) of pyrrole 17a, thus giving rise to the2,2′-bispyrrole 18. Intramolecular palladium (II)-promoted2,2′-bispyrrole coupling of 18 using stoichiometric, polymer-supportedpalladium (II) acetate (2-3% Pd, 1% cross-linked polystyrene) affords19, a key precursor on the synthetic pathway to 14. Selectivetransformation of the propionate side chain into a trimethylamino propylside chain followed by conversion of the remaining carbomethoxy groupinto the required aldehyde 20, sets the stage for condensation withpyrrole 21 leading to direct formation of 22. Transformation of 22 intoits pyrromethane-BF₂ complex, as described above, and subsequentconversion of the acid functionality into the required aldehydegenerates 14. There is ample precedent in the work of Lugtenburg (Vos deWael, E., et al., (1977), Recl. Trav. Chim. Pays-Bas 96, 306-309),suggesting that only the desired pyrromethane-BF₂ complex will form.

The preparation of engineered dye C shown in Scheme 3, necessitatescoupling of carboxylic acid succinimidyl ester 1 with commerciallyavailable L-Cys(SO₃+Na⁻)—OH leading to acid 23. Esterification with theknown protected ethanolamine 24 (Powell, J., et al., (1986) LithiumAluminum Hydride Reductions; A New Hydrolysis Method for IntractableProducts, Synthesis Communications 338-340) provides, after cleavage ofthe TBS group and oxidation of the resultant alcohol 25, carboxylic acid26. The conversion of 26 into sulfosuccinimidyl ester dye C is carriedout as detailed above.

The elaboration of D is realized by conversion of carboxylic acid 15into its corresponding sulfosuccinimidyl ester. Following the protocoldetailed above for the conversion of 1 into C leads to D.

The synthesis of E (Scheme 4) requires coupling of the carboxylic acidsulfosuccinimidyl ester 27, derived from 23, with 24 followed by thecleavage (TBAF, HOAc, THF) of the silyl protecting group and subsequentconversion (TsCl, pyr, NaI, acetone) of the alcohol into iodide 28.Alkylation of the phenoxide anion derived from 32 with iodide 28 givesrise to 33. Completion of the synthesis of E requires 1) reduction(NaBH₄) of the methyl ketone functionality, 2) coupling of the resultantalcohol 34 with the new reagent 38 leading to 39 and 3) brief exposureof 39 to trimethyl silyl iodide, which leads, upon aqueous workup, to E.The required aromatic piece 32 is prepared from commercially availableacetovanillone 29, as outlined in Scheme 5, using the protocol ofÅkerblorn (Akerblom, E. B., et al., (1998) Six new photolabile linkersfor solid-phase synthesis. 1. Methods of preparation. Mol. Divers., 3,137-148). The novel reagent 38 is prepared from the commerciallyavailable sulfo-NHS acetate 35 as detailed in Scheme 6. The methylationof sulfonate anions is well documented in the literature (Trujillo, J.L. and Gopalan, A. S. (2000) Facile Esterfication of Sulfonic Acids andCarboxylic Acids with Triethylorthoacetate, Tetrahedron Letters 34,7355-7358), as well as the treatment of N-hydroxysuccinimide withbis(trichloromethyl) carbonate (Konakahara, T., et al., (1993) AConvenient Method for the Synthesis of Activated N-Methylcarbamates,Synthesis 103-106).

The construction of F commences with carboxylic acid 16 and employs thesame protocol that is detailed above for the synthesis of E.

The synthesis of the thienyl boradiazaindacene G (Scheme 7) requiressynthesis of the pyrrole 42 from the bromopyrrole 5 via chemistrydescribed for the synthesis of 8. Coupling of 42 with 2-bromothiopheneleads to the thienyl pyrrole 43, which upon formylation produces 44.Coupling of 44 with pyrrole 21 affords 45, which upon conversion of theacid functionality into the required aldehyde and subsequent exposure toborontrifluoride etherate gives rise to 46. The transformation of 46into G utlizes the protocol outlined above for the conversion of 14 intodye B.

The preparation of H (Scheme 8) requires coupling of the formyl pyrrole8, prepared as detailed above, with pyrrole 47, whose synthesis isdescribed below. The coupled material 48 is converted as detailed aboveinto the difluoroboradiazaindacene 49. The transformation of 49 into Hfollows the protocol discussed above for the preparation of C with theminor modification that a quaternized lysine residue is incorporatedinto the peptide chain. Pyrrole 47 is synthesized from the known pyrrole50 (Muchowski, J. M. and Hess, P., (1988) Tetrahedron Lett., 29(26),3215) as illustrated in Scheme 8. Selective reduction of the morereactive ester followed by protection of the resultant hydroxyl as asilyl ether followed by straightforward transformation of the remainingester into a formyl group provides 51. Chain extension via an Emmonsreaction followed by reduction of the olefin generates 52. Protection ofthe pyrrole nitrogen followed by sequential cleavage (TBAF) of the silylether, a Finkelstein reaction (MsCl; NaI acetone) and displacement withpotassium thioacetate affords 53. Exposure of 53 to K₂CO₃/MeOH gives wayto the corresponding thiol which upon oxidation, methylation, cleavageof the BOC group and hydrolysis of the esters provides 47.

Construction of the dye I requires the preparation ofdifluoroboradiazaindacene 56 which is subjected to the protocol detailedabove for the synthesis of dye D. Once again a minor modification of thescheme is required to incorporate the quaternized lysine. The formationof 56, as detailed in Scheme 9 requires condensation of pyrrole 20 withpyrrole 54 to produce 55. Cleavage of the silyl group, oxidation to thealdehyde and introduction of the difluorobora unit result in 56.

Pyrrole 54 is prepared from 50. Selective deprotection of the mostreactif benzyl ester, reduction to the alcohol and protection as a TBSgroup yields to pyrrole 57. Conversion of 57 into 58 is done using thechemistry described above for the conversion of 52 into 53. Exposure of58 to K₂CO₃/MeOH gives way to the corresponding thiol which uponoxidation, methylation and cleavage of the BOC group provides 59.Finally, hydrogenolysis of the benzyl ester, reduction of the resultingacid to the alcohol, protection of the alcohol functionality (TBS) andhydrolysis of the methyl sulfonate result in 54.

EXAMPLE 2 Synthesis of Dyes A2-I2

This examples sets forth an example of synthesis for dyes A2-12 (FIGS.9A and 9B)

The series A2-I2 presents 2 major differences with respect to the seriesA-I shown in Example 1. These two modifications are exemplified with thesynthesis of A2 in Scheme 10. The first one is the replacement of thecysteic acid residue with arginine in the conversion of 60 to 61. Usingarginine in place of cysteic acid in the various synthetic routes willeasily take care of this modification. The second difference is in thereplacement of the side chain containing the quaternary ammonium groupwith a sulfonate. This can be carried out by using isopropyldiethylphosphorylmethanesulfonate in a Horner-Wadsworth-Emmons couplingstep as in the conversion of 6 into 62 (Carretero J. C., Demillequand M.and Ghosez L, Synthesis of alpha, beta-unsaturated sulphonates via theWittig-Horner reaction; Tetrahedron Vol. 43,No. 21, pp. 5125-5134,1987.). The newly introduced ethyl sulfonate is then deprotected to thesulfonate following the formylation step to generate 63. 63 is convertedinto 60 using the procedure described for the synthesis of A. Thesesteps can be generalized to the synthetic routes of dyes B2-I2.

In addition to the two previously described variations, the dyes H2 andI2 present a third modification with respect to dyes H and I: ashortening of the sulfonate side chain from a three to a two carbontether. This adjustment is made by substituting pyrroles 68 and 71 topyrroles 47 and 54 respectively in the synthesess of H and I. Thesyntheses of fragments 68 and 71 are illustrated in Scheme 11.

The synthesis of Pyrrole 68 starts with the knownpyrrole-3-carboxaldehyde 4. (Bray, B. L., et al.,“N-(Triisopropylsilyl)pyrrole. A Progenitor “Par Excellence” of3-Substituted Pyrroles”, J. Organic Chem. 1990, 55(26),6317-6328)Coupling of 4 with the known isopropyl diethylphosphorylmethanesulfonate(Carretero J, et al.; Tetrahedron Vol. 43,No. 21, pp. 5125-5134, 1987)and subsequent catalytic hydrogenation of the resulting olefin leads tointermediate 66. Formylation of 66 into 67 is carried out under theVilsmeier-Haack conditions. At this point the stage is set for theDoebner coupling of formyl pyrrole 67 with mono ethyl malonate whichafter hydrogenolysis and hydrolysis of the esters will generate 68.

The synthesis of 71 starts with the known ester 69. (“Sambrotta, L., etal., “Synthesis of 8-Demethyl-8-Formyl Protoporphyrin IX and of8-Demethyl Protoporphyrin IX”, Tetrahedron 1989, 45, 6645-6652)Treatment of 69 under the conditions described above leads to ester 70,which is subsequently reduced to the corresponding alcohol, hydrolysedand selectively reprotected to yield 71.

EXAMPLE 3 Synthesis of Dyes A3-I3

This Examples Sets Forth an Example of Synthesis for Dyes A3-I3 (FIGS.10A and 10B)

Dyes A3-I3 are synthesized as described in Example 2 for Dyes A2-I2except that the arginine residue is substituted with a trimethylatedlysine, using trimethylated lysine in place of arginine in the varioussynthetic routes. Trimethyllysine has an advantage for some applicationsthat it is not cleaved by trypsin, whereas arginine is, in general,cleaved by trypsin. Arginine is not a problem with many applications ofthe zwitterionic dyes described, where the dyes are removed afterprotein separation and quantitation, but before protease digestion formass spectral analysis.

Additional examples of synthesis for dyes A-I, A2-I2 and A3-I3 are foundin U.S. patent application Ser. No. 10/623,447, hereby incorporated byreference.

EXAMPLE 4 Evaluation and Optimization of Labeling of Target Proteinsfrom Different Types of Samples

The sensitivity of labeling to pH, buffer type, and common salts in thereaction medium is tested for different sample types, using parallelreadout of the results of different conditions on 1D electrophoresis andquantitation of labeled proteins with laser excited fluorescent gelscanning. Phosphate buffer is used near pH 7.4, a phosphate/boratemixture near pH 8, and borate near pH 8.5 or 9.0. Tris buffers or otherbuffers with potentially reactive amines must be avoided. The best ratioof labeling to hydrolysis is near pH 8.5, unless SDS or other anionicdetergent is used to solubilize the proteins and then a somewhat higherpH is favorable. The labeling rate of amino groups with thesulfo-succinamidyl or succinamidyl groups increases with pH, however attoo high a pH the succinamidyl group hydrolyzes. Labeling kinetics aremeasured by quenching the labeling reactions at different times withexcess glycine, hydroxylamine or low pH. Possible enhancement oflabeling can be assessed for different samples in the presence of thedetergents, urea, and thiourea used for IEF, using, 1D SDS gels andfluorescence emission as the readout.

After favorable pH and labeling times are established for samples fromdifferent organisms or tissues, experiments may be carried out to varythe optical labeling molecule/protein ratio during labeling. Theapproximate number of optical labeling molecules per labeled protein andthe relative fluorescence of the optical labeling molecules on differentlabeled proteins is determined, using on-line fluorescence andabsorbance detection in HPLC gel filtration experiments. The HPLC gelfiltration separates the free optical labeling molecule from the labeledproteins. Proteins used in such studies can be chosen to allowseparation based on size by HPLC gel filtration. The amount of eachprotein added to the reaction mixture is known and the amount of 280absorbance observed from the known amount of protein is determined inthe HPLC on unlabeled and labeled samples. The stoichiometry of theoptical labeling molecule to protein is determined from absorbancemeasurements of the dye moiety of the optical labeling on each proteinpeak and the relative extinction coefficients of the protein and the dyemoiety. Fluorescence/absorbance ratios on each protein peak, relative tothe free optical labeling molecule, allows detection of fluorescencequenching by the protein or by excessive numbers of optical labelingmolecule/protein.

Such experiments also allow determination of the ratio of proteinlabeling to optical labeling molecule hydrolysis under differentconditions, as it is desirable to minimize the remaining free opticallabeling molecule for improved detection of low molecular weightproteins. The ratio of hydrolyzed and unreacted optical labelingmolecule are determined on the free optical labeling molecule fractionby RP-HPLC. Too high an optical labeling molecule concentration duringlabeling might produce some dye fluorescence quenching by excessiveprotein labeling or produce inactive optical labeling molecule dimers oreven higher multimers from these particular optical labeling molecule.If optical labeling molecule dimerization occurs, it will be controlledby variation of labeling conditions. If necessary, moresterically-hindered tertiary amine groups (such as a t-butyl) can besubstituted for the titratable group in the synthesis of the dye.

The strength of on-gel fluorescent signals is measured as a function ofthe number of optical labeling molecules per protein using gelfiltration analysis of aliquots of the samples, where the labelingstoichiometry has been determined by gel filtration, as described above,it is not anticipated that the quenching of fluorescent signals willdiffer much in solution vs. in gels, as a function of the number ofoptical labeling molecule/protein, except at the highest proteinloadings on gels where fluorescence quenching may be observed. Suchexperiments establish the range of linearity of fluorescence signals andthe dynamic range of detection of optical labeling molecule-labeledproteins on gels. Any differences in labeling of proteins in specificmixtures of proteins with different members of the optical labelingmolecule sets, or families, can be detected by splitting identicalprotein mixtures, labeling each half of the sample with differentoptical labeling molecule, mixing the samples and detecting thefluorescence ratios for each band on 2D gels. Any departure from aconstant ratio of fluorescence signals across bands on the gel wouldindicate differences in labeling, but this is not expected to besignificant. If significant optical labeling molecule-dependent labelingis seen with some proteins, a labeling reversal experiment should bedone routinely to allow correction for this effect in practicalfunctional proteomics experiments.

The stability of the dye binding to the labeled proteins can bedetermined by centrifugal filtration to concentrate each protein peakfrom HPLC gel filtration, incubation of the purified, labeled proteinsfor various times (in the presence of sodium azide and proteaseinhibitors) and measuring any loss of labeling by rerunning on gelfiltration. The UV-reversible linkages in some of the compounds requireprotection from fluorescent light for highest stability, and sampletubes must be foil wrapped and manipulated under dim incandescent light.

EXAMPLE 5 Effect of Optical Labeling Molecule on Protein Solubility andTwo-Dimensional Gel Electrophoresis Mobility

The effect of the optical labeling molecule on protein solubility and2DE mobility is assessed using fluorescent signals and radioactivelabeling of standard proteins. The solubilities of labeled proteins canbe assessed by running them on IEF (isoelectric focusing) and 2D(two-dimensional) electrophoresis to assess any changes of retention ofproteins on the IEF strips before and after labeling. Retention ofprotein on the IEF strips and poor transfer into the second dimension isoften found in 2D electrophoresis if sample loadings are too high or ifsolubilization conditions are inadequate. Fluorescent signals of labeledproteins retained on IEF strips provide semi-quantitative measurementsof limited solubility since the strong signals can exceed the linearrange. The use of the optical labeling molecules of the invention willlead to substantial protein solubility increases compared to theunlabeled protein samples. To verify this phenomenon, radioactivelylabeled standard proteins and complex mixtures of proteins from cellsare used for assessment of any labeling induced gel mobility shifts (seebelow) and these same radioactive proteins will be useful forquantitative solubility assessments. Phosphorimaging of the 2DE gels,and any protein residues on the IEF strips, provides a quantitativemeasure of insoluble proteins remaining on the LEE strips, relative tothe radioactivity on the second dimension.

Two methods of radioactive labeling of the standard proteins are used.N-acetyl labeling with tritiated acetic anhydride at near neutral pHlargely couple to N-terminal groups. Excess acetic anhydride will beremoved by HPLC gel filtration, followed by fluorescent dye labeling ofthe epsilon amino groups of lysine at elevated pH (e.g. 8.5). Analternative method of radioactive labeling first reduces proteinsulfhydryl groups with tributylphosphine (TBP), tricarboxyethylphosphine (TCEP), or other trisubstituted phosphine compound. Thesulfhydryl groups are then labeled with radioactive iodoacetamide andthe amino groups labeled with dyes.

2D gels are run on the radioactively tagged and fluorescently labeledproteins after low (substoichiometric), medium (one or two opticallabeling molecules per protein) and high optical labeling moleculeslabeling (many optical labeling molecules per protein). Gels are scannedfor fluorescence and the location of radioactive spots will be measuredby phosphorimaging on the same BioRadFX Fluorescent Gel Scanner andPhosphoimager. The radioactivity shows the position of proteins that arenot labeled, as well as the labeled proteins. Thus, any optical labelingmolecule-induced shifts in protein patterns is detected and monitored bycomparing radioactivity patterns to fluorescence patterns. An expectedreduction of shifts is assessed using the optical labeling moleculeswith titratable groups. The dyes with titratable amine groups areespecially valuable in the high pH range from 10-12. Commercial IEFstrips are now available from Pharmacia up to pH=11 and if strips up topH=12 are not commercially available, the needed strips may be preparedfollowing publications of the Gorg lab in Munich (Gorg, A., et al.,(1999) Recent developments in two-dimensional gel electrophoresis withimmobilized pH gradients: wide pH gradients up to pH 12, longerseparation distances and simplified procedures, Electrophoresis 20:712-717; Gorg, A. (1999) IPG-Dalt of very alkaline proteins, MethodsMol. Biol. 112, 197-209; Gorg, A., et al., (2000) The current state oftwo-dimensional electrophoresis with immobilized pH gradients,Electrophoresis 21, 1037-1053, each of which is hereby expresslyincorporated by reference). The larger the multiplicity of opticallabeling molecules labeling on target proteins the larger thefluorescent signals (up to the point where fluorescence quenchingbecomes a problem). Thus, the labeling conditions can be optimized formaximum sensitivity consistent with acceptable mobility shifts formixtures of proteins from particular organisms or tissues.

With two (or multiple) color ratio recording of fluorescent signals, theinformation content as to which proteins are changing in level withphysiological stimulus is insensitive to optical labelingmolecule-induced shifts as long as the shifts are the same or verysimilar for the different dyes. However, increased complexity or spotdistortion would occur if labeling shifted the gel mobility withincreasing number of optical labeling molecules bound/protein. Iflabeled protein spots are resolved from other proteins then thefluorescence ratios will still contain reliable information on therelative expression of proteins under different physiologicalconditions. Thus, any significant shifts with labeling will favorincreased reliance on narrow pH range IEF gels to spread proteins over 1or 2 unit pH range. Optical labeling molecule-induced shifts are notexpected to be very large due to the modest resolution of 2D gels. Atradeoff between minimum complexity and lower sensitivity withsub-stoichiometric labeling, to possibly more spot complexity andhighest sensitivity with high optical labeling molecule labeling will beunder experimental control.

EXAMPLE 6 Testing of the Protein Pre-Labeling Methods on StandardProteins

A very large range of protein abundance/concentration is found in cells,tissues and bodily fluids. Increased dynamic range of proteinmeasurement can be obtained by labeling samples at more than one levelof dye multiplicity and scanning gels at several differentphotomultiplier amplifications. After the desirable conditions fordifferent multiplicity of optical labeling molecule labeling areestablished for particular protein mixtures, the detection limit andlinearity of the fluorescence signal vs. amount of protein loading canbe determined. These experiments can be carried out at low labelingmultiplicity, medium multiplicity and high multiplicity of opticallabeling molecule labeling that is found to be useful in priorexperiments and can also determine the dynamic range for the method andthe scanner in practice. A dilution series of standard proteins labeledwith the optical labeling molecules is made and the different dilutionsrun on different lanes of ID gels.

Similar experiments can be carried out with two and three or severaldifferent optical labeling molecules using identical standard proteinmixtures. In multiple color optical labeling molecule experiments, dyecross talk and multiplex sensitivity is determined, using constantamounts of one or two of the labeled protein mixtures (at a relativelyhigh level) and varying the amount of proteins labeled with a second orthird optical labeling molecule in steps from the detection limit tovery high levels. The degree of crosstalk between the two main groups ofoptical labeling molecule investigated is extremely low due to theessentially non-existent direct excitation of the partner dyes by thelasers to be used. Double-label pairs with minimum cross-talk are dyesA, C, E, or H (excited with the 488 nm laser)-paired with B, D, F or I(excited with the 633 nm laser).

Dye G can be used as a third optical labeling molecule and excited withthe 532 mm laser, with only modest cross talk expected with the otherdyes. The degree of crosstalk is determined by comparing gels from astandard curve of protein fluorescence on a dilution series, using asingle optical labeling molecule, to the same dilution series in thepresence of a constant, high level of proteins labeled with a secondoptical labeling molecule. Any preference of optical labeling moleculefor different proteins is determined by labeling protein mixturesseparately with the different optical labeling molecules, mixing the twoor three different labeled proteins in the same amounts, runningelectrophoretic separations and determining the fluorescence colorratios.

EXAMPLE 7 Recovery of Proteins from 2D Gels and Efficiency of Removal ofOptical Labeling Molecule

The recovery of proteins from 2D gels and efficiency of removal of theoptical labeling molecule is assessed and optimized using radioactivelylabeled proteins with and without the optical labeling molecule. Initialexperiments are carried out in aqueous solution on glycine-quenched dyesto test the amount and type of UV irradiation needed to remove thereversible linker efficiently, using RP-FPLC to analyze the products.Known amounts of labeled standard proteins are run in duplicates.Fluorescence and phosphoimager scanning can be used to confirm thedilution series. Consistent-sized gel circles are punched out of thegel, frozen in liquid nitrogen and the gel pieces powdered with astainless steel rod in microfuge tubes. One of the duplicate samples iscounted for radioactivity and the other is freeze-dried and thenrehydrated in a buffer containing Promega autolysis-resistant trypsin,(+/−TCEP and IAA to enhance recovery of cysteine-containing peptides).Dye labeled and control samples are treated with UV (365 nm mercurylamp) to remove the reversible optical label molecule linkage. Afterincubation (24-48 hours) gel pieces are extracted with 50% acetonitrileand the supernatant harvested by centrifugal filtration using a filterthat is resistant to acetonitrile (e.g. Millipore Biomax) to retain thegel fragments. The extraction is repeated once or more with acetonitrileand the extracts are counted to determine the recovery of peptideradioactivity. Control proteins with no labels are hydrolyzed insolution with trypsin in H2O¹⁸ to mark the trypsin cleavage sites with0¹⁸ substitution (Shevchenko, A. and Shevchenko, A. (2001) Evaluation ofthe Efficiency of 1n-gel Digestion of Proteins by Peptide IsotopicLabeling and MALDI Mass Spectrometry. Anal. Biochem 296, 279-283, herebyexpressly incorporated by reference). Aliquots of the 0¹⁸-labeledpeptides are added to the extraction steps and the ratios of 0¹⁶peptides to 0¹⁸ peptides monitored by mass spectrometry to determine thepercentage of recovery of peptides from the protein. The peptides arerun on MALDI and ESI/MS/MS to determine peptide recovery +/−UV treatmentto remove the dye labels, using 0¹⁸ internal standards. Standardacrylamide gels and meltable Proto-Preps system gels (NationalDiagnostics) will be compared. Protocols for efficient protein digestionand peptide recovery will be optimized to maximize the conditions foreffective protein identification using mass spectral analysis. 0.1%octyl glucoside may be included to improve recovery of tryptic peptidesfrom in-gel digests (Mann, M., et al., (2001) Analysis of proteins andproteomes by mass spectrometry, Annu. Rev. Biochem. 10, 437-473, herebyexpressly incorporated by reference).

EXAMPLE 8 Testing of the Optical Labeling Molecules on Total BacterialProteins

The properties of the optical labeling molecules i can be evaluated onthe complex protein mixture in the total protein complement of anorganism. For example, the hyperthermophilic archeabacterium, Sulfolobussolfararicus, can be use to evaluate the optical labeling molecules.

An advantage to the use of a microorganism for testing and evaluation ofproteomic methodology is that all the proteins in the microorganisms caneasily be radioactively labeled, using radioactive sulfur⁻³⁵ in thegrowth medium. Radioactive labeling provides tremendous advantages forassessment of protein recovery from gels and any label-induced gelmobility shifts. Essentially the same techniques are used for analysisof the total Sulfolobus proteins as was described above. Sulfolobusprovides a wide range (about 3,316 proteins in the geonome) of proteinswith a much greater variety of characteristics, than possessed bystandard protein mixtures (discussed in earlier sections). Inparticular, there is the opportunity to discover any dye-specificlabeling preferences in the wide range of Sulfolobus proteins usingsimple dye cross-over labeling experiments. Comparison of radioactivityand dye labeling are used to detect any dye labeling-induced shifts oncomplex protein mixtures from Sulfolobus. Protein spots are cut out ofthe gel, the dye label is removed by UV irradiation (365 or 308 nm), theproteins digested with trypsin in the presence of octyl glucoside toenhance recovery (Katayama, H., et al., (2001) Improvement of in-geldigestion protocol for peptide mass fingerprinting by matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry, RapidComm. Mass Spectrom. 15, 1416-1421, hereby expressly incorporated byreference), peptides are extracted and submitted to mass spectralanalysis using the best procedures available (Gygi, S. P. and Aebersold,R. (2000) Mass spectrometry and proteomics, Curr. Opin. Chem. Biol. 4:489-494; Loo, J. A., et al., (1999) High sensitivity mass spectrometricmethods for obtaining intact molecular weights from gel-separatedproteins, Electrophoresis, 20, 743-748, Kraft, P. et al., (2001) Massspectrometric analysis of cyanogen bromide fragments of integralmembrane proteins at the picomole level: application to rhodopsin, Anal.Biochem. 292, 76-86, each of which is hereby expressly incorporated byreference). For example, nano-spray and tandem mass spectral techniquescan be used as a method to identify proteins and post-translationalmodifications.

EXAMPLE 9 Multiplex Detection of Phosphorylation

Phosphorylation is one of the most common post-translationalmodifications in cellular regulation, but because of the labile natureof this modification, phosphorylation is difficult to detect by massspectrometry. Some of the Trk receptor isoforms are phosphorylated andthere is evidence that several signaling cascades are activated(Patapoutian A, and Reichardt L F., Trk receptors: mediators ofneurotrophin action, Curr Opin Neurobiol. 2001 Jun. 11 (3):272-80,hereby expressly incorporated by reference). In addition to the methodsof detecting the presence or absence of proteins, or quantity ofprotein, with fluorescence detection, multiplex detection ofphosphorylation can be performed with all the proteins on the samesample as described previously and below.

The dorsal root ganglia (DRG) cells are cultured as described (Garner,A. S, and Large, T. H. (1994) Isoforms of the avian TrkC receptor: anovel kinase insertion dissociates transformation and process outgrowthfrom survival, Neuron 13, 457-472), unstimulated cells are labeled with³³P phosphate and growth factor stimulated cells are labeled with ³²Pphosphate. After suitable incubation the two cell samples are extracted.The ³³P-labeled extracts are reacted with a first optical labelingmolecule and the ³²P-labeled extracts are reacted with a seconddifferent optical labeling molecule. The first and the second opticallabeling molecules are chosen from the same set of optical labelingmolecules so that the optical signal is different but the physicalcharacteristics are similar. The labeled extracts will be mixedtogether, run on 2D gels and laser scanned for the protein expressionratios between the stimulated and unstimulated cells. In addition, twophosphoimager image plates will be exposed simultaneously on two sidesof the same gel, one phosphoimager plate directly on the gel and theother having a I mil thickness of copper foil in front of tinephosphoimager plate (Bossinger, J., et al., (1979) Quantitative analysisof two-dimensional electrophoretograms, J. Biol. Chem., 254, 7986-7998;Johnston, R. F., et al., (1990) Autoradiography using storage phosphortechnology, Electrophoresis, 11, 355-360; Pickett, S. C., et al., (1991)Quantitative double-label autoradiography using storage phosphorimaging, Molecular Dynamics Application Note). The directly exposed P1plate registers the sum of both isotopes, whereas the copperfoil-filtered phosphoimager image almost entirely blocks the ³′P,whereas barely attenuating the signals from the ³²P. The results ofthese studies will be compared to direct dye staining of the serine andthreonine phosphorylated proteins using beta-elimination of thephosphates by base treatment of the gels after fluorescent andphosphoimager scanning or after transfer of proteins to PVDF membranesand staining of the beta-eliminated sites with high sensitivityfluorescent dyes, as shown in FIGS. 7A and 7B and discussed above.

Thus, the multiplex methods of the invention can be extended for withsimultaneous monitoring of changes in phosphorylation, as well as thechanges in the level and postranslational modification of the proteinsassociated with function.

1. An optical labeling molecule for labeling a target analytecomprising: a. a boron difluoride diaza-indacene-propionic acid (BODIPY)zwitterionic dye moiety comprising at least one positive or at least onenegative charge moiety added to obtain a net neutral charge, wherein thepositive charge moiety is selected from a group consisting of aquaternary ammonium group and a guanidinium group, wherein the positivecharge moiety is not titratable between the pH of 3-12, and wherein thenegative charge moiety is selected from the group consisting of asulfonate group and a sulfate group, wherein the negative charge moietyis not titratable between the pH of 3-12; b. a functional linker moiety;and c. a titratable group moiety, wherein the titratable group is atertiary amine which closely approximates the pK of the group removedfrom the target analyte by reaction with the functional linker.
 2. Theoptical labeling molecule of claim 1, wherein the zwitterionic dyemoiety comprises a chromophore responsible for a detectable opticalsignal.
 3. The optical labeling molecule of claim 1, wherein the saidpositive and said negative charge moieties are added to a neutral dye ora charged dye to produce a zwitterionic dye molecule.
 4. The opticallabeling molecule of claim 1, wherein the zwitterionic dye moietycomprises at least one quaternary ammonium group and at least onesulfonate group.
 5. The optical labeling molecule of claim 1, whereinthe zwitterionic dye moiety comprises at least two quaternary ammoniumgroups or at least two guanidinium groups and at least two sulfonategroups.
 6. The optical labeling molecule of claim 1, further comprisinga cleavable moiety selected from a group consisting of a chemicalmoiety, a photocleavable moiety and an enzymatically cleavable moiety.7. The optical labeling molecule of claim 6, wherein the photocleavablemoiety is selected from a group consisting of an O-nitrobenzyliccompound, a benzoin moiety, and a nitrophenylcarbamate ester.
 8. Theoptical labeling molecule of claim 1, further comprising a second label,wherein the second label comprises a light stable isotope or a heavystable isotope.
 9. The optical labeling molecule of claim 1, wherein thelinker moiety is an amino group reactive linker selected from the groupconsisting of an imidoester, a N-hydroxysuccinimidyl ester, anisothiocyanate, a sulfosuccinimidyl group, an aldehyde, and asulfonylchloride-reactive linker and a sulfhydryl-reactive linker. 10.The optical labeling molecule of claim 9, wherein thesulfhydryl-reactive linker is selected from the group consisting of amaleimide, an iodoacetamide, an alkyl bromide and a benzoxidiazole. 11.The optical labeling molecule of claim 1, wherein the dye moiety furthercomprising at least one added negative charge moiety selected from asulfonate group or a sulfate group and at least one added positivecharge moiety selected from a group consisting of a quaternary ammoniumgroup, a guanidinium group and a positive charge group.
 12. The opticallabeling molecule of claim 1, wherein the labeling molecule has thegeneral structure: wherein ZD is the zwitterionic dye moiety, T is thetitratable moiety, and A is the linker moiety.
 13. The optical labelingmolecule of claim 1, wherein the labeling molecule has the generalstructure: ZD-T-A- wherein ZD is the zwitterionic dye moiety, T is thetitratable moiety, and A is the linker moiety.
 14. The optical labelingmolecule of claim 1, wherein the labeling molecule has the generalstructure: T-ZD-C-A- or ZD-T-C-A wherein ZD is the zwitterionic dyemoiety, T is the titratable moiety, C is the cleavable moiety, and A isthe linker moiety.
 15. The optical labeling molecule of claim 1, whereinthe labeling molecule has the general structure: T-ZD-C-I-A- wherein ZDis the zwitterionic dye moiety, T is the titratable moiety, C is thecleavable moiety, I is the stable isotope moiety, and A is the linkermoiety.
 16. The optical labeling molecule of claim 1, wherein thelabeling molecule has the general structure: ZD-T-C-I-A- wherein ZD isthe zwitterionic dye moiety, T is the titratable moiety, C is thecleavable moiety, I is the stable isotope moiety, and A is the linkermoiety.
 17. The optical labeling molecule of claim 16, furthercomprising: (d) a photocleavable moiety wherein such photocleavablemoiety is an O-nitrobenzylic compound.
 18. The optical labeling moleculeof claim 1 comprising: a functional linker moiety wherein such linker isa N-hydroxysuccinimidyl ester.
 19. A cellular component selected fromthe group consisting of proteins, carbohydrates, lipids, and nucleicacids covalently attached to the optical labeling molecule of claim 1.20. The cellular component of claim 19, wherein the said component is aprotein.
 21. The cellular component of claim 19, wherein the saidcomponent is a carbohydrate.
 22. The optical labeling molecule of claim1, prepared by (a) providing a boron difluoride diaza-indacene-propionicacid (BODIPY) dye moiety comprising one or more positive or one or morenegative charge moieties; (b) adding at least one positive charge moietyselected from the group consisting of a quaternary ammonium group, aguanidinium group and a positive charge group, wherein the positivecharge moiety is not titratable between the pH of 3-I2, or at least onenegative charge moiety selected from a group consisting of a sulfonategroup and a sulfate group, wherein the negative charge group is nottitratable between the pH of 3-I2 to form a zwitterionic dye moiety; (c)contacting the zwitterionic dye moiety with a titratable group moiety;and (d) contacting the product from step (c) with a functional linkermoiety to provide a optical labeling molecule comprising a zwitterionicdye moiety characterized by a net neutral charge when bound to thetarget analyte.
 23. The optical labeling molecule of claim 22, whereinthe zwitterionic dye moiety is a fluorescent dye.
 24. The opticallabeling molecule having formula (I):


25. The optical labeling molecule which is selected from the groupconsisting of: